During the current outbreak of Zika virus (ZIKV), there has been an increased association of ZIKV infections with microcephaly and other congenital neurological disorders prompting extensive efforts to develop antiviral strategies; however, none have thus far been approved for use in humans. The identification of host cellular factors required for virus replication may lead to novel therapeutic strategies. The host ubiquitin (Ub) system is a conserved cellular pathway important in many functions, including innate immune signaling and virus replication. The goal of this proposal is to define the molecular mechanisms of ZIKV replication involving the ubiquitin system, and identify host proviral factors that can be targeted for therapeutic intervention. The envelope (E) protein of flaviviruses is essential for virus replication because it mediates attachment to host cell receptors, thereby promoting virus internalization. Further, the E protein may also confer specific cellular tropism by recognizing host factors expressed by specific cell types. Although the E proteins of closely related flaviviruses, like dengue virus (DENV), are well characterized, little is known about ZIKV-E function, and whether host cellular factors are linked to the characteristic neurological disorders observed during ZIKV infections is unknown. There is a major gap in knowledge regarding how ZIKV establishes productive infections in its host and whether it uses the ubiquitin system for replication. Because of its critical function in virus internalization, the E protein has been proposed as a target for therapeutic intervention. Therefore, the identification of host cellular factors required for virus replication by regulating E protein function may provide novel antiviral targets. Our preliminary data indicate that the ZIKV-E protein is ubiquitinated by TRIM7, a member of the E3-Ub ligase tripartite motif (TRIM) family. Knockdown of TRIM7 resulted in reduced ubiquitination of E, which correlated with reduced ZIKV replication in placenta-derived cell lines, suggesting that TRIM7 has a proviral function by ubiquitinating E. TRIM7 is highly expressed in placenta and reproductive tissues, two important sites of ZIKV replication. Therefore, our hypothesis is that the envelope protein of ZIKV is ubiquitinated by TRIM7 to promote ZIKV replication within specific sites of the host. By using in vitro biochemical approaches, recombinant ZIKV mutant viruses, and an in vivo mouse model, we will determine whether ubiquitination of E is important for ZIKV tropism and reveal how TRIM7 contributes to ZIKV replication. We propose the following specific aims: 1) Determine the role of ubiquitination of the ZIKV envelope protein in virus replication and 2) Determine the molecular mechanism by which TRIM7 regulates ZIKV replication. The outcome of these studies is significant because it will provide fundamental knowledge about the mechanism of ZIKV replication, which may apply to other members of the Flaviviridae family. This work also identifies a host cellular factor, TRIM7, that can potentially be targeted as a novel therapeutic strategy.